A hallmark of infection with positive strand RNA viruses is the impressive intracellular membrane rearrangements induced within host cells to facilitate replication of their viral genomes. This project will use a multidisciplinary approach to investigate the molecular mechanisms that drive intracellular membrane rearrangements during flavivirus infection. The focus of this project will be on dengue virus, an NIAID category A priority pathogen, because of its medical importance and the substantial data and reagents we and others have accumulated over the last several years. Dengue virus causes about 50-100 million infections per year making it the most important arbovirus worldwide. It belongs to the flavivirus genus, which comprises more than 70 members, many of which are important human pathogens. Infection of cells with dengue virus results in enhanced synthesis of new lipid-derived structures and extensive membrane rearrangements. It has been shown that these membranous structures are required for a productive dengue virus infection, however, the molecular requirements that underlie the formation of these structures remains largely unknown. This project will determine the membrane composition of these structures and the cellular signaling cascades and trafficking pathways recruited by dengue virus for their formation and function. A high-resolution mass spectrometry based approach has been developed to initially identify the lipid-mediators important for the formation of these intracellular membranous structures. Preliminary data from these analyses have indicated a clear difference between the overall lipid composition of dengue virus infected cells compared to uninfected cells. These observations will be extended to determine how these changes in lipid profiles translate to the observed ultrastructural modifications in dengue virus infected cells. High-resolution mass spectrometry analyses will be employed to identify the differentially expressed lipids. Specifically isolated membrane fractions will be analyzed from infected cells and used to determine the temporal changes in lipid profile and their accumulation in these structures during the course of infection. Using lifetime imaging technology and confocal microscopy, the temporal redistribution of key lipids into these membranous structures will be visualized and these will be correlated with the various stages of the viral life cycle including viral gene expression, viral RNA replication and virus assembly. Using molecular genetics we will define the viral gene products responsible for these rearrangements. Ultimately, the experiments proposed should provide a framework for understanding the virus-induced alterations in lipid metabolism and ultrastructure in dengue virus infected cells and provide the basis for future experiments detailing the lipid-mediated molecular interactions between virus and host. PUBLIC HEALTH RELEVANCE: The flaviviruses represent a large and significant set of viral pathogens that infect hundreds of millions of people each year. By discovering how dengue virus changes the lipid components of cells, it may be possible to better understand how the virus causes disease, and may give insight into how to design novel therapeutics that modify these changes to inhibit the virus.